Methods for Treating Cancer

ABSTRACT

The invention relates to novel peptides having an HDM-2 targeting sequence that target the human minute binding protein-2. The invention also relates to fusion proteins comprising a HDM-2 targeting sequence. The invention also relates to methods of using the peptides to treat cancer.

PRIORITY

This application is a divisional application of U.S. Ser. No.15/706,942, filed Sep. 18, 2017, which is a divisional application ofU.S. Ser. No. 15/231,839, filed Aug. 9, 2016, (U.S. Pat. No. 9,765,117)which claims priority to Provisional Application U.S. Ser. No.62/209,182, filed Aug. 24, 2015, each of which is hereby incorporated byreference in its entirety.

A sequence listing is provided herewith.

BACKGROUND

The p53 protein is a cell cycle regulatory protein. It functions toinhibit the oncogenic effects of a number of oncogene proteins thatinduce mitosis by blocking transcription of proteins that induce mitosisand by inducing the transcription of proteins that block mitosis, andpromote apoptosis. There is a correlation between the absence of the p53protein and cell transformation and malignant disease. Haffner, R &Oren, M. (1995) Curr. Opin. Genet. Dev. 5: 84-90.

The p53 protein consists of 393 amino acids and binds to anotherimportant regulatory protein, the MDM-2 protein. HDM-2 (human doubleminute) and MDM-2 (mouse double minute) are p53 specific E3 ubiquitinprotein ligases that suppresses the transcriptional activity of thetumor suppressor p53 and promote its degradation thereby limiting thetumor suppressor function of p53. HDM-2 and MDM-2 induce ubiquitinationof p53 and target it for proteolysis in the proteasome (Alarcon-Vargaset al. 2002 Carcinogenesis 23: 541-547). Each of HDM-2 and MDM-2 have ap53 binding domain.

The MDM-gene that encodes the MDM-2 protein is a known oncogene. TheMDM-2 protein forms a complex with the p53 protein, which results in thedegradation of the p53 protein by a ubiquination pathway. The p53protein binds to MDM-2 protein using an amino acid sequence thatincludes residues 12-29 of the p53 protein (Haffner, R & Oren, M. (1995)Curr. Opin. Genet. Dev. 5: 84-90).

Overexpression or amplification of MDM-2 (HDM-2) protein has been foundin 40-60% of human malignancies, including 50% of human breast tumors.It has been suggested that the anti-tumor effect of the p53 proteinmight be enhanced by peptides capable of interfering with the binding ofthe MDM-2 protein to the p53 protein. A number of investigators havesuggested that the MDM-2/p53 complex might be a target for rational drugdesign. See, e.g., Christine Wasylyk et al., “p53 Mediated Death ofCells Overexpressing MDM2 by an Inhibitor of MDM2 Interaction with p53”,Oncogene, 18, 1921-34 (1999), and U.S. Pat. No. 5,770,377 to Picksley etal. Cancers that have been shown to have an increased level of membraneassociated HDM-2 include TUC-3 pancreatic cells, MIA-PaCa-2 humanpancreatic cancer cells, MCF-7 human breast cancer cells, A-2058 humanmelanoma cells (Sarafraz-Yazdi, E. et al. 2010 Proc Natl Acad Sci USA107(5): 1918-1923), K562 leukemia cells (Davitt et al. 2014 Annals ofClinical and Laboratory Science 44(3): 241-248) and primary humanovarian cancers (Sarafraz-Yazdi et al. 2015, Annals of Clinical andLaboratory Science, in press).

U.S. Pat. Nos. 8,822,419, 7,531,515, 7,883,888, 7,745,405, US2011/0183915 and US 2014/0371156 are incorporated by reference.

SUMMARY OF THE INVENTION

The invention relates to novel peptides that target the human minutebinding protein-2 (HDM-2) comprising an HDM-2-targeting amino acidsequence, as well as fusion peptides comprising this sequence, and theuse of a peptide according to the invention for treatment of cancer.

The invention relates to a peptide comprising the amino acid sequence

(SEQ ID NO: 2) PPLSQTSFAEYWNLLSP.Preferably, the peptide further comprises a membrane penetrating aminoacid sequence, which peptide has increased cellular uptake, and whichpreferably forms the carboxy terminal sequence of said peptide. Inpreferred embodiments, the membrane penetrating sequence comprises theamino acid sequence:

(SEQ ID NO: 4) KKWKMRRNQFWVKVQRG.

The invention also relates to a peptide comprising the amino acidsequence:

(SEQ ID NO: 3) H-Pro-Pro-Leu-Ser-Gln-Thr-Ser-Phe-Ala-Glu-Tyr-Trp-Asn-Leu-Leu-Ser-Pro-Lys-Lys-Trp-Lys-Met-Arg-Arg-Asn-Gln-Phe-Trp-Val-Lys-Val-Gln-Arg-Gly-OH.

The invention also relates to a peptide having a length of 8 amino acidsto 35 amino acids, comprising a sequence consisting of the amino acidsequence PPLSQTSFAEYWNLLSP (SEQ ID NO: 2). The invention also relates toan HDM-2-targeting peptide comprising the amino acid sequence: X₁ X₂ X₃X₄ X₅ TSX₆AEYWNLLSP (SEQ ID NO 26) wherein X₁,X₆, independently, may beany amino acid, provided the fusion peptide causes death of cells whichover-express HDM-2.

The invention also relates to a fusion peptide sequence comprising SEQID NO: 26 and another sequence comprising a cell penetrating peptide.

A fusion peptide according to the invention may also include one or morecytotoxic components, for example, a toxin, a drug, a radionuclide, anantibody or antibody fragment and combinations thereof.

In preferred embodiments, an alpha helix stabilizing amino acid residuemay be included at either or both the amino or carboxyl terminal ends ofthe HDM-2 targeting peptide or of the HDM-2 targeting portion of afusion peptide. Amino acids that stabilize the alpha helix include Leuand Glu, (particularly on the amino terminal end of the helix), and Metand Phe. Therefore, in a preferred embodiment the amino terminus of theHDM-2 targeting peptide or the HDM-2 targeting portion of a fusionpeptide comprises Leu, Glu, Met or Phe. In another preferred embodiment,the carboxy terminus of the HDM-2 targeting peptide or the HDM-2targeting portion of a fusion peptide comprises Leu, Glu, Met or Phe.

Preferably, the HDM-2 targeting peptide or portion of a fusion peptideincludes an amino acid sequence substantially identical toPPLSQETFSDLWKLL (SEQ ID NO. 1), which includes residues 12-26 of humanp53 protein, and differs from the naturally occurring p53 sequence asfollows: E at position 6 is substituted with T, T at position 7 issubstituted with S, S at position 9 is substituted with A, D at position10 is substituted with E, L at position 11 is substituted with Y and Kat position 13 is substituted with N, and including two additional aminoacids, S and P, at the carboxy terminus.

A peptide according to the invention may be in the form of a compositioncomprising the peptide, and/or a pharmaceutical composition comprisingthe peptide, in combination with a pharmaceutically acceptable carrier,and/or in the form of a kit comprising the peptide and packagingmaterials.

The invention also pertains to a method of treating cancer in a subject,comprising administering to the subject a therapeutically effectiveamount of a composition comprising a peptide of the invention; a methodof selectively necrosing cells, comprising providing a plurality ofcells, including at least one cancer cells and at least one normal cell,administering to the cells a composition wherein the compositioncomprises the peptide, and wherein said composition results inmembranolysis of said cancer cells, but does not affect said normalcells; and to a method of causing membranolysis in cancer cells,comprising administering to at least one cancer cell a compositioncomprising the peptide.

In one embodiment, of a method of selectively necrosing cells or amethod of causing membranolysis, membranolysis indicates necrosis of atleast one cancer cell. The method can also include a step of measuringin a cell medium contacted with the cells a level of LDH, whereby anincrease in the level of LDH in the cell medium after administering thecompound as compared to the level of LDH in the cell medium prior toadministering the compound indicates necrosis of at least one cancercell. The method can also comprise the step of microscopy of the cellswhereby morphology in the cells treated with the compound that isidentical to untreated cells indicates the absence of necrosis. Thecompound can be administered at a dosage between 20 μmol/ml and 160μmol/ml.

The method of treating cancer may further comprise the step of observingin a medium of the cancer cells an early release of LDH, observingmembranolysis of the cancer cells, observing a decrease from a number ofpre-treatment cancer cells to a number of post treatment cancer cells;observing tumor cell eradication; repeating the administering step untila result is reached; observing necrosis in the cancer cells; and orobserving a non-response in the normal cell, wherein the non-responseindicates the normal cell is unaffected.

DETAILED DESCRIPTION OF THE INVENTION

As used herein, “peptide” means at least 5 amino acids. In one aspect, apeptide is 5-35 amino acids, for example, 5, 6, 7, 8, 9, 10, 15, 20, 25,30 or 35 amino acids. In another embodiment, a peptide is 8-30 aminoacids (for example, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25or 30 amino acids). In another embodiment, a peptide is 8-20 aminoacids. A peptide of the invention comprises at least 5 amino acidresidues of residues 12-26 of the human p53 protein.

A peptide according to the invention can include D-amino acids,non-peptide or pseudo-peptide linkages and peptidyl mimics. In addition,the peptide and peptide mimics can be modified, e.g. glycosylated ormethylated. Synthetic mimics of targeting peptides are also included.

The peptides of the invention can be used for treating a samplecomprising both healthy, normal cells and cancer cells, including celllines, tissue samples, tumors, and/or a subject diagnosed with cancerand in need of treatment.

The present invention provides methods of treating cancer using a fusionpeptide as set forth herein.

“Cancer” refers to a group of diseases involving abnormal cell growthincluding but not limited to solid tissue tumor and non-solid tissuetumor cancers, for example, astrocytoma, bladder cancer, brain cancer,breast cancer, cervical cancer, colorectal cancer, endometrial cancer,gastric cancer, lung cancer, melanoma, leukemia, lymphoma, ovariancancer, pancreatic cancer, prostate cancer, renal carcinoma, sarcoma,thyroid cancer, glioblastoma, multiple myeloma, myelodysplasticsyndrome, mesothelioma, acute myeloid leukemia, childhood leukemia,chronic myeloid leukemia, myelodysplastic syndrome, Hodgkin's lymphoma,and Polycythemia Vera.

A fusion peptide according to the invention comprises a portion which isa transmembrane penetrating sequence or membrane resident peptide (MRP).MRP is required for induction of necrosis by the inventive peptides.Expression of the p53 sequence in the absence of the MRP in cancer cellscauses wild-type p53-dependent apoptosis, or programmed cell death andnot tumor cell necrosis.

In one embodiment the MRP includes predominantly positively chargedamino acid residues since a positively charged leader sequence maystabilize the alpha helix of a subject peptide. Examples of MRPs whichmay be useful to the HDM-2 targeting compounds of the present inventionare described in Futaki, S. et al (2001) Arginine-Rich Peptides, J.Biol. Chem. 276, 5836-5840, and include but are not limited to thefollowing MRPs in Table 1, below. The MRP may be, for example, thepeptides identified as SEQ ID NOs: 4-25 in Table 1. For some of theMRPs, the numbering of the amino acid residues making up the MRP isindicated in parentheses.

TABLE 1 SEQ ID NO: SEQUENCE NAME SEQ ID PPLSQTSFAEYWNLLSP SLH-1 NO: 3KKWKMRRNQFWVKVQRG SEQ ID KKWKMRRNQFWVKVQRG Membrane resident NO: 4peptide (MRP), reverseomer of Antennapedia SEQ ID YGRKKRRQRRRPPQHIV-1 TAT(47-60), NO: 5 membrane resident peptide SEQ ID GRKKRRQRRRPPQD-TAT, membrane NO: 6 resident peptide SEQ ID GAAAAAAAAAPPQR-TAT G(R)₉PPQ, NO: 7 membrane resident peptide SEQ ID PKKKRKVSV40-NLS, membrane NO: 8 resident peptide SEQ ID KRPAAIKKAGQAKKKKnucleoplasmin-NLS, NO: 9 membrane resident peptide SEQ IDTRQARRNRRRRWRERQR HIV REV (34-50), NO: 10 membrane resident peptideSEQ ID RRRRNRTRRNRRRVR FHV (35-49) coat, NO: 11 membrane residentpeptide SEQ ID KMTRAQRRAAARRNRWTAR BMV GAG (7-25), NO: 12membrane resident peptide SEQ ID TRRQRTRRARRNR HTLV-II REX 4-16, NO: 13membrane resident peptide SEQ ID KLTRAQRRAAARKNKRNTR CCMV GAG (7-25),NO: 14 membrane resident peptide SEQ ID NAKTRRHERRRKLAIER P22 N (14-30),NO: 15 membrane resident peptide SEQ ID MDAQTRRRERRAEKQAQWLAMBDA N(1-22), NO: 16 KAAN membrane resident peptide SEQ IDTAKTRYKARRAELIAERR Phi N (12-29), NO: 17 membrane resident  peptideSEQ ID TRRNKRNRIQEQLNRK YEAST PRP6 (129- NO: 18 124), membraneresident peptide SEQ ID SQMTRQARRLYV HUMAN U2AF, NO: 19membrane resident peptide SEQ ID KRRIRRERNKMAAAKSR HUMAN C-FOS (139-NO: 20 NRRRELTDT 164), membrane resident peptide SEQ IDRIKAERKRMRNRIAASKS HUMAN C-JUN (252- NO: 21 RKRKLERIAR 279), membraneresident peptide SEQ ID KRARNTEAARRSRARKLQRMKQ YEAST GCN4, NO: 22membrane resident peptide SEQ ID KLALKLALKALKAALKLA Example membraneNO: 23 resident peptide (MRP) SEQ ID LLIILRRRIRKQAKAHSK p-vec, membraneNO: 24 resident peptide SEQ ID RRRRRRRR (Arg)8 or any  NO: 25poly-R from  (R)₄-(R)₁₆,   membrane resident peptide

Additional MRPs useful according to the invention are described e.g., inScheller et al. (2000) Eur. J. Biochem. 267:6043-6049, and Elmquist etal., (2001) Exp. Cell Res. 269:237-244, the contents of which areincorporated herein by reference in its entirety.

The positively charged MRP may include the amino acid sequence:

(SEQ ID NO: 4) KKWKMRRNQFWVKVQRG.which is related to the reverseomer sequence of the antennapediasequence. The MRP can be attached to the carboxyl terminal end of apeptide of the invention (e.g. peptide) or to the amino terminal end ofa peptide of the invention.

The invention also provides for amino acid insertional derivatives ofthe invention that include amino and/or carboxyl terminal fusions aswell as intra-sequence insertions of single or multiple amino acids.Insertional amino acid sequence variants are those in which one or moreamino acid residues are introduced into a predetermined site in asubject peptide although random insertion is also possible with suitablescreening of the resulting product. Deletional variants may be made byremoving one or more amino acids from the sequence of a subject peptide.Substitutional amino acid variants are those in which at least oneresidue in the sequence has been removed and a different residueinserted in its place.

When the synthetic peptide is derivatised by amino acid substitution,the amino acids are generally replaced by other amino acids having likeproperties such as hydrophobicity, hydrophilicity, electronegativety,bulky side chains and the like. As used herein, the terms “derivative”,“analogue”, “fragment”, “portion” and “like molecule” refer to a subjectpeptide having the amino acid sequence as set forth in SEQ ID NOs: 2 or3, having an amino acid substitution, insertion, addition, or deletion,as long as said derivative, analogue, fragment, portion, or likemolecule retains the ability to enter and selectively kill transformedor neoplastic cells.

In certain embodiments, the compounds and compositions of the presentinvention have a three dimensional shape or conformation in analpha-helix-loop-alpha-helix. Interaction of the composition with thecancer cell membrane is facilitated by the alpha-helix-loop-helixconformation.

Also contemplated are peptides having a degree of rigidity than isgreater than that of the synthetic peptide SLH-1. Thealpha-helix-loop-alpha-helix, that results in an amphipathic structure,in which hydrophobic amino acid residues occupy one face of the moleculewhile polar residues occupy the opposite face of the molecule, is onepossible conformation of the molecule. A number of membrane-activepeptides, such as melittin and magainin, have these required structuresthat result in cell membrane lysis though not with the same specificityas SLH-1. Thus, if agents can be administered to a peptide-basedcomposition to increase the rigidity, or if a non-peptide, called apeptidomimetic, rigid molecules of similar size, with a similaramphipathic structure, may be employed with the present invention, thenthe conformation will more immediately affect the cancer cells. Rosal R,Brandt-Rauf P W, Pincus M R, Wang H, Mao Y, Fine R L. The role ofalpha-helical structure in p53 peptides as a determinant for theirmechanism of cell death: necrosis versus apoptosis. Adv Drug Deliv Rev2005; 57:653-60; Pincus, M. R. (2001) “The Physiological Structure andFunction of Proteins” in Principles of Cell Physiology (Chapter 2),Third Edition, Ed., N. Sperelakis, Academic Press, New York, pp. 19-42;3. Dathe, M. and Wieprecht, T. (1999) Structural Features of HelicalAnti-Microbial Peptides: Their Potential to Modulate Activity on ModelMembranes and Biological Cells. Biochem. Biochem. Biophys. Acta 1462,71-87.

In certain embodiments, the peptide of the invention is relativelysmall. It is more likely that large peptide, non-peptide, andcombination peptide/non-peptide compositions will cause a significantimmunologic response. Thus, the immune system of the subject beingtreated is less likely to trigger an immune response against smallmolecules, i.e. peptides of <35AA than large molecule compositions,i.e., proteins with >35AA. Preferably, the synthetic peptide materialsof the present invention are on the order of about thirty-five (35)amino acids or fewer, for example, 35, 30, 25, 20, 19, 18, 17, 16, 15,14, 13, 12, 11, 10, 9 or 8.

In another embodiment, the composition of the invention has a longhalf-life and remains in the body for longer periods of time beforedecomposing. A composition with a longer half-life may have an increasedlongevity, allowing it to be transported through the body to kill morecancer cells or treat cancers located in different parts of the organismupon a single administration. The invention provides for peptides,including SLH-1, modified to include a D-amino acid on the aminoterminal end in order to slow peptidase activity of the molecule. Inanother embodiment, the peptidase inhibitor leupeptin may be attached tothe carboxyl terminal end of SLH-1 to slow peptidase activity andlengthen the half-life of the molecules.

One or more of the methods referenced herein may optionally include areiteration or repeated administration step. That is, after theadministration step, it is possible to determine whether a plurality ofsubsequent cancer cells exist and remain intact. If so, it is possibleto complete one or more of the method steps for each of the methodspreviously discussed, including the administration of the compound,HDM-2 recognition agent, and the like.

The compounds, agents, and materials used in conjunction with one ormore of the aforementioned methods are desirably in a purified form.Purified form, as used herein, generally refers to material which hasbeen isolated under certain desirable conditions that reduce oreliminate unrelated materials, i.e. contaminants. Substantially freefrom contaminants generally refers to free from contaminants withinanalytical testing and administration of the material. Preferably,purified material is substantially free of contaminants is at least 50%pure, more preferably, at least 90% pure, and more preferably still atleast 99% pure. Purity can be evaluated by conventional means, e.g.chromatography, gel electrophoresis, immunoassay, composition analysis,biological assay, NMR, and other methods known in the art.

At least one cancer cell, as used herein, may similarly refer to aplurality of cancer cells. A plurality of cells may include, a sample ofcells, a tissue sample, a tumor, and/or even a subject having cancer. Atleast one cell may refer to one cell, a plurality of cells, a sample ofcells, a tissue sample, and/or even a subject. A plurality of cellsincluding at least one cancer cell and at least one non-cancerous cellmay refer to a mixture of cells in a sample, an area of tissue includingboth cancerous and non-cancerous tissues, and or a subject diagnosedwith cancer.

The term “subject”, as used herein may refer to a patient or patientpopulation diagnosed with, or at risk of developing one or more forms ofcancer. Also, as used herein, a subject may refer to a living animal,including mammals, in which cancer may be induced throughtransplantation or xenotransplanting which may be subsequently treatedwith the methods and compounds of the present invention or which havedeveloped cancer and need veterinary treatment. Such subjects mayinclude mammals, for example, laboratory animals, such as mice, rats,and other rodents; monkeys, baboons, and other primates, etc. They mayalso include household pets or other animals in need of treatments forcancer.

The terms “therapeutically effective dosage” and “effective amount”refer to an amount sufficient to kill one or more cancer cells. Atherapeutic response may be any response that a user (e.g. a clinicianwill recognize) exhibits as an effective response to the therapy,including the foregoing symptoms and surrogate clinical markers. Thus, atherapeutic response will generally be an amelioration or inhibition ofone or more symptoms of a disease or disorder, e.g. cancer.

Administering, as referred to by one or more of the methods of thepresent invention, may include contacting. The term “contacting” refersto directly or indirectly bringing the cell and the compound together inphysical proximity. The contacting may be performed in vitro or in vivo.For example, the cell may be contacted with the compound by deliveringthe compound into the cell through known techniques, such asmicroinjection into the tumor directly, injecting the compound into thebloodstream of a mammal, and incubating the cell in a medium thatincludes the compound.

Any method known to those in the art for contacting a cell, organ ortissue with a compound may be employed. Suitable methods include invitro, ex vivo, or in vivo methods. In vitro methods typically includecultured samples. For example, a cell can be placed in a reservoir(e.g., tissue culture dish), and incubated with a compound underappropriate conditions suitable for inducing necrosis in cancer cells.Suitable incubation conditions can be readily determined by thoseskilled in the art.

Ex vivo methods typically include cells, organs or tissues removed froma mammal, such as a human. The cells, organs or tissues can, forexample, be incubated with the compound under appropriate conditions.The contacted cells, organs or tissues are normally returned to thedonor, placed in a recipient, or stored for future use. Thus, thecompound is generally in a pharmaceutically acceptable carrier.

In vivo methods are typically limited to the administration of acompound, such as those described above, to a mammal, preferably ahuman. The compounds useful in the methods of the present invention areadministered to a mammal in an amount effective in necrosing cancercells for treating cancer in a mammal. The effective amount isdetermined during pre-clinical trials and clinical trials by methodsfamiliar to physicians and clinicians.

The compounds useful in the methods of the invention may also beadministered to mammals by sustained release, as is known in the art.Sustained release administration is a method of drug delivery to achievea certain level of the drug over a particular period of time. The leveltypically is measured by serum or plasma concentration.

The compounds of one or more of the aforementioned methods of thepresent invention may be administered to a human in an amount effectivein achieving its purpose. The effective amount of the compound to beadministered can be readily determined by those skilled in the art, forexample, during pre-clinical trials and clinical trials, by methodsfamiliar to physicians and clinicians. Typical daily doses includeapproximately 1 mg to 1000 mg.

An effective amount of a compound useful in the methods of the presentinvention, preferably in a pharmaceutical composition, may beadministered to a mammal in need thereof by any of a number ofwell-known methods for administering pharmaceutical compounds. Thecompound may be administered systemically or locally.

Any formulation known in the art of pharmacy is suitable foradministration of the compounds useful in the methods of the presentinvention. For oral administration, liquid or solid formulations may beused. Some examples of formulations include tablets, capsules, such asgelatin capsules, pills, troches, elixirs, suspensions, syrups, wafers,chewing gum and the like. The compounds can be mixed with a suitablepharmaceutical carrier (vehicle) or excipient as understood bypractitioners in the art. Examples of carriers and excipients includestarch, milk, sugar, certain types of clay, gelatin, lactic acid,stearic acid or salts thereof, including magnesium or calcium stearate,talc, vegetable fats or oils, gums and glycols.

Formulations of the compounds useful in the methods of the presentinventions may utilize conventional diluents, carriers, or excipientsetc., such as those known in the art to deliver the compounds. Forexample, the formulations may comprise one or more of the following: astabilizer, a surfactant, preferably a nonionic surfactant, andoptionally a salt and/or a buffering agent. The compound may bedelivered in the form of an aqueous solution, or in a lyophilized form.Similarly, salts or buffering agents may be used with the compound.

The compounds of the present invention may be administered intherapeutically effective concentrations, to be provided to a subject instandard formulations, and may include any pharmaceutically acceptableadditives, such as excipients, lubricants, diluents, flavorants,colorants, buffers, and disintegrants. Standard formulations are wellknown in the art. See, e.g. Remington's pharmaceutical Sciences, 20thedition, Mach Publishing Company, 2000. The formulation may be producedin useful dosage units for administration by any route that will permitthe compound to contact the cancer cell membranes. Exemplary routes ofadministration include oral, parenteral, transmucosal, intranasal,insulfation, or transdermal routes. Parenteral routes includeintravenous, intra-arterial, intramuscular, intradermal, subcutaneous,intraperitoneal, intraductal, intraventricular, intrathecal, andintracranial administrations.

The pharmaceutical forms suitable for injection include sterile aqueoussolutions or dispersions and sterile powders for the extemporaneouspreparation of sterile injectable solutions or dispersions. The ultimatesolution form in all cases must be sterile and fluid. Typical carriersinclude a solvent or dispersion medium containing, e.g., water bufferedaqueous solutions, i.e., biocompatible buffers, ethanol, polyols such asglycerol, propylene glycol, polyethylene glycol, suitable mixturesthereof, surfactants or vegetable oils. Sterilization may beaccomplished utilizing any art-recognized technique, including but notlimited to filtration or addition of antibacterial or antifungal agents.

The compounds of the present invention may be administered as a solid orliquid oral dosage form, e.g. tablet, capsule, or liquid preparation.The compounds may also be administered by injection, as a bolusinjection or as a continuous infusion. The compounds may also beadministered as a depot preparation, as by implantation or byintramuscular injection.

The compounds, agents, and materials referenced in the present inventionmay be in a “pharmaceutically acceptable carrier”. A pharmaceuticallyacceptable carrier includes any and all solvents, dispersion media,coatings, antibacterial and antifungal agents, isotonic agents and thelike. The use of such media and agents are well-known in the art. Thephase ‘pharmaceutically acceptable’ refers to molecular entities andcompositions that are physiologically tolerable and do not typicallyproduce unwanted reactions when administered to a subject, particularlyhumans. Preferably, as used herein, the term “pharmaceuticallyacceptable” means approved by a regulatory agency of the federal or astate government or listed in the U.S. Pharmacopeia or other generallyrecognized pharmacopeia for use in animals, and more particularly, inhumans. The term carrier refers to a diluent, adjuvant, excipient orvehicle with which the compounds may be administered to facilitatedelivery. Such pharmaceutical carriers can be sterile liquids, such aswater and oils, or organic compounds. Water or aqueous saline solutions,and aqueous dextrose and glycerol solutions are preferably employed ascarriers, particularly as injectable solutions.

The synthetic peptides which may include the compounds, agents, andmaterials used with the present methods of the present invention may besynthesized by a number of known techniques. For example, the peptidesmay be prepared using the solid-phase technique initially described byMerrifield (1963) in J. Am. Chem. Soc. 85:2149-2154. Other peptidesynthesis techniques may be found in M. Bodanszky et al. PeptideSynthesis, John Wiley and Sons, 2d Ed., (1976) and other referencesreadily available to those skilled in the art. A summary of polypeptidesynthesis techniques may be found in J. Stuart and J. S. Young, SolidPhase Peptide Synthesis, Pierce Chemical Company, Rockford, Ill.,(1984). Peptides may also be synthesized by solid phase or solutionmethods as described in The Proteins, Vol. II, 3d Ed., Neurath, H. etal., Eds., pp. 105-237, Academic Press, New York, N.Y. (1976).Appropriate protective groups for use in different peptide syntheses aredescribed in the texts listed above as well as in J. F. W. McOmie,Protective Groups in Organic Chemistry, Plenum Press, New York, N.Y.(1973). The peptides of the present invention may also be prepared bychemical or enzymatic cleavage from larger portions of the p53 proteinor from the full length p53 protein. Likewise, membrane-residentsequences for use in the synthetic peptides of the present invention maybe prepared by chemical or enzymatic cleavage from larger portions orthe full length proteins from which such leader sequences are derived.

Additionally, the peptides of the present invention may also be preparedby recombinant DNA techniques. For most amino acids used to buildproteins, more than one coding nucleotide triplet (codon) can code for aparticular amino acid residue. This property of the genetic code isknown as redundancy. Therefore, a number of different nucleotidesequences may code for a particular subject peptide selectively lethalto malignant and transformed mammalian cells. The present invention alsocontemplates a deoxyribonucleic acid (DNA) molecule that defines a genecoding for, i.e., capable of expressing a subject peptide or a chimericpeptide from which a peptide of the present invention may beenzymatically or chemically cleaved.

The synthetic peptides of the present invention may be synthesized by anumber of known techniques. For example, the peptides may be preparedusing the solid-phase technique initially described by Merrifield (1963)in J. Am. Chem. Soc. 85:2149-2154. Other peptide synthesis techniquesmay be found in M. Bodanszky et al. Peptide Synthesis, John Wiley andSons, 2d Ed., (1976) and other references readily available to thoseskilled in the art. A summary of polypeptide synthesis techniques may befound in J. Sturart and J. S. Young, Solid Phase Peptide Synthesis,Pierce Chemical Company, Rockford, Ill., (1984). Peptides may also besynthesized by solution methods as described in The Proteins, Vol. II,3d Ed., Neurath, H. et al., Eds., pp. 105-237, Academic Press, New York,N.Y. (1976). Appropriate protective groups for use in different peptidesyntheses are described in the texts listed above as well as in J. F. W.McOmie, Protective Groups in Organic Chemistry, Plenum Press, New York,N.Y. (1973). The peptides of the present invention may also be preparedby chemical or enzymatic cleavage from larger portions of the p53protein or from the full length p53 protein. Likewise, leader sequencesfor use in the synthetic peptides of the present invention may beprepared by chemical or enzymatic cleavage from larger portions or thefull length proteins from which such leader sequences are derived.

Additionally, the peptides of the present invention may also be preparedby recombinant DNA techniques. For most amino acids used to buildproteins, more than one coding nucleotide triplet (codon) can code for aparticular amino acid residue. This property of the genetic code isknown as redundancy. Therefore, a number of different nucleotidesequences may code for a particular subject peptide selectively lethalto malignant and transformed mammalian cells. The present invention alsocontemplates a deoxyribonucleic acid (DNA) molecule that defines a genecoding for, i.e., capable of expressing a subject peptide or a chimericpeptide from which a peptide of the present invention may beenzymatically or chemically cleaved.

A peptide of the invention includes a peptide that is a substantiallysimilar variant of SEQ ID Nos 1, 2, 3 and/or 4. A peptide of theinvention includes a peptide that is a substantially similar variant ofamino acid residues of the HDM-2 binding region of p53. A peptide of theinvention includes a peptide that is a substantially similar variant ofamino acid residues 12-26 of the p53 protein and include the amino acidsequence NLLSP.

A “substantially similar variant” may be at least 25% identical to apeptide of the invention, as long as it is able to target HDM-2 on thesurface of cells, and/or provided it causes cell death. Of course, thepercent identity can be higher, e.g., 30%, 35, 40%, 45%, 50%, 55%, 60%,65%, 67%, 69%, 70%, 73%, 75%, 77%, 83%, 85%, 87%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, or 99% identity. In general, the substitutionsare conservative substitutions.

The variants thereof can also have substitutions, deletions oradditions. Alterations may produce conservative or non-conservativeamino acid substitutions, deletions or additions. In some embodimentsthe substitution, deletion or insertion is of 1, 2, 3, 4, 5, 6, 7, 8, 9or more amino acids. The skilled artisan is fully aware of amino acidsubstitutions that are either less likely or not likely to significantlyaffect protein function (e.g., replacing one aliphatic amino acid with asecond aliphatic amino acid). Guidance concerning how to makephenotypically silent amino acid substitutions is provided in Bowie, J.U. et al., “Deciphering the Message in Protein Sequences: Tolerance toAmino Acid Substitutions,” Science 247:1306-1310 (1990).

The peptides or the variants thereof can also have modified backbones,e.g., oligocarbamate or oligourea backbones (see, e.g., Wang et al., J.Am. Chem. Soc. 119:6444-6445 (1997); Tamilarasu et al., J. Am. Chem.Soc. 121:1597-1598 (1999); Tamilarasu et al., Bioorg. Of Med. Chem.Lett. 11:505-507 (2001)).

Sequence identity may be determined by sequence comparison and alignmentalgorithms known in the art. To determine the percent identity of twoamino acid sequences, the sequences are aligned for optimal comparisonpurposes (e.g., gaps can be introduced in the first sequence or secondsequence for optimal alignment). The amino acid residues atcorresponding amino acid positions are then compared. When a position inthe first sequence is occupied by the same residue as the correspondingposition in the second sequence, then the molecules are identical atthat position. The percent identity between the two sequences is afunction of the number of identical positions shared by the sequences(i.e., % homology=# of identical positions/total # of positions times100), optionally penalizing the score for the number of gaps introducedand/or length of gaps introduced.

The comparison of sequences and determination of percent identitybetween two sequences can be accomplished using a mathematicalalgorithm. In one embodiment, the alignment generated over a certainportion of the sequence aligned having sufficient identity but not overportions having low degree of identity (i.e., a local alignment). Apreferred, non-limiting example of a local alignment algorithm utilizedfor the comparison of sequences is the algorithm of Karlin and Altschul(1990) Proc. Natl. Acad. Sci. USA 87:2264-68, modified as in Karlin andAltschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-77. Such an algorithmis incorporated into the BLAST programs (version 2.0) of Altschul, etal. (1990) J. Mol. Biol. 215:403-10.

In another embodiment, the alignment is optimized by introducingappropriate gaps and percent identity is determined over the length ofthe aligned sequences (i.e., a gapped alignment). To obtain gappedalignments for comparison purposes, Gapped BLAST can be utilized asdescribed in Altschul et al., (1997) Nucleic Acids Res.25(17):3389-3402. In another embodiment, the alignment is optimized byintroducing appropriate gaps and percent identity is determined over theentire length of the sequences aligned (i.e., a global alignment). Apreferred, non-limiting example of a mathematical algorithm utilized forthe global comparison of sequences is the algorithm of Myers and Miller,CABIOS (1989). Such an algorithm is incorporated into the ALIGN program(version 2.0) which is part of the GCG sequence alignment softwarepackage. When utilizing the ALIGN program for comparing amino acidsequences, a PAM120 weight residue table, a gap length penalty of 12,and a gap penalty of 4 can be used.

Also provided are nucleic acid sequences encoding each of SEQ ID NOs:1-4.

The nucleic acid sequence corresponding to SEQ ID NO: 1 is:

(SEQ ID NO: 38) ccc cct ctg agt cag gaa aca ttt tca gac cta tggaaa cta cct.

The nucleic acid sequence corresponding to any one of SEQ ID NOs: 2-4includes a sequence wherein each of the amino acids of SEQ ID NO: 2-4 isencoded by any of the corresponding DNA codons presented in Table 2.

TABLE 2 Amino Acid SLC DNA codons Isoleucine I ATT, ATC, ATA Leucine LCTT, CTC, CTA, CTG, TTA, TTG Valine V GTT, GTC, GTA, GTG Phenylalanine FTTT, TTC Methionine M ATG Cysteine C TGT, TGC Alanine A GCT, GCC, GCA,GCG Glycine G GGT, GGC, GGA, GGG Proline P CCT, CCC, CCA, CCG ThreonineT ACT, ACC, ACA, ACG Serine S TCT, TCC, TCA, TCG, AGT, AGC Tyrosine YTAT, TAC Tryptophan W TGG Glutamine Q CAA, CAG Asparagine N AAT, AACHistidine H CAT, CAC Glutamic acid E GAA, GAG Aspartic acid D GAT, GACLysine K AAA, AAG Arginine R CGT, CGC, CGA, CGG, AGA, AGG Stop codonsStop TAA, TAG, TGA

A representative nucleic acid sequence corresponding to SEQ ID NO: 2 is:

(SEQ ID NO: 39) ccc cct ctg agt cag act tct ttt gct gaa tat tggaat cta ctt tct cct

A representative nucleic acid sequence corresponding to SEQ ID NO: 3 is:

(SEQ ID NO: 40) ccc cct ctg agt cag act tct ttt gct gaa tat tggaat cta ctt tct cct aaa aaa tgg aaa atg cgc cgcaac cag ttt tgg gtg aaa gtg cag cgc ggc.

A representative nucleic acid sequence encoding SEQ ID NO: 4 is:

(SEQ ID NO: 41) aaa aaa tgg aaa atg cgc cgc aac cag ttt tgg gtgaaa gtg cag cgc ggc.

Variations of each of SEQ ID NOs: 2-4 can occur due to redundancies inthe genetic code.

Peptides

SLH-1,H-Pro-Pro-Leu-Ser-Gln-Thr-Ser-Phe-Ala-Glu-Tyr-Trp-Asn-Leu-Leu-Ser-Pro-Lys-Lys-Trp-Lys-Met-Arg-Arg-Asn-Gln-Phe-Trp-Val-Lys-Val-GlnArg-Gly-OH (SEQ ID NO: 2) (1 g), are synthesized using solid phasemethods (Shaanxi Zhongbang Pharma-Tech Corp., NanguanZhengjie, Xi'an,China) and was >95% pure by HPLC and mass spectrographic analysis. Theitalicized sequence corresponds to the MRP segment that allows entry ofthe whole peptide into cells. A negative control peptide, PNC-29,containing the X13 peptide from cytochrome P450 (bold) attached to theMRP (italics),H-Met-Pro-Phe-Ser-Thr-Gly-Lys-Arg-Ile-Met-Leu-Gly-Glu-Lys-Lys-Trp-Lys-Met-Arg-Arg-Asn-Gln-Phe-Trp-Val-Lys-Val-Gln-Arg-Gly-OH(SEQ ID NO: 27), is likewise synthesized by solid phase methods and islikewise>95% pure. The X13 peptide replaces the p53 sequence of SLH-1.

FITC- and TAMRA-Double Fluorophore-Labeled SLH-1 Peptide

This peptide is synthesized at the Biopeptide Corp., La Jolla, Calif.,with two fluorescent labels: amino terminal 5,6-carboxy-fluorescein(green fluorescence) and carboxyl terminal 5-tetramethyl Rhodamine(TAMRA) (red fluorescence) and assessed for purity.

Plasmids

DNA encoding the modified human p53 amino acid sequence of SLH-1, iscloned into the mammalian pTracer-SV40 (green-fluorescent protein[GFP]-expressing) expression vector downstream to the SV40 promoter.This vector constitutively expresses a cloned gene (Invitrogen,Carlsbad, Calif.). Also included in the vector is another expressioncassette which is linked in tandem to the SV40-p53 amino acid-expressingunit. The second expression cassette contains a CMV promoter driving theexpression of the GFP-Zeocin resistance gene fusion protein. The vectoris used to transform TOP10F′ chemically competent E. coli using methodsof transformation known in the art, and plated on Zeocin-containing agarplates for overnight growth. Eight colonies are then used to inoculatecultures in Low Salt Luria Broth (1% bacterial tryptone, 0.5% yeastextract, 0.5% NaCl, and 25 m/ml Zeocin). Cultures are grown underconstant shaking at 200 rpm for 16 h in a 37° C. incubator and plasmidsare extracted using a Qiagen Spin Miniprep Kit.

Construct sense and anti-sense strands of the cDNA encoding the p53sequence of SLH-1 are synthesized. For maximum protein translation intransfected cell lines, the start codon is placed within a Kozaksequence, i.e., GCCACCATGG (SEQ ID NO: 28) (with ATG being the startcodon), which is the optimal context for initiation of translation invertebrate mRNA. The strands (250 nmol/ml) are annealed in annealingbuffer by heating to 95° C., and then cooling to room temperature. Theannealed double stranded sequence-encoding cDNA corresponding to the p53sequence of SLH-1 is then digested with NotI and EcoRI simultaneously.For example, a total of 20 μg of pTracer-SV40 is digested with 60 unitsof NotI and 60 units of EcoRI. Double-digested pTracer-SV40 and cDNAencoding the p53 sequence of SLH-1 are then electrophoresed through 0.8%and 2.5% agarose gel, respectively. Gel bands containing DNA ofappropriate size are excised, and DNA content is extracted using theNucleoTrap Gel Extraction kit (ClonTech, Mountain View, Calif.).Purified vector and cDNA are ligated with T4 ligase (for 12 hr, at 4°C.) (New England Biolab, Ipswich, Mass.). Two μl of ligation reaction isthen dispensed into a vial containing 50 μl One Shot TOP10F′ competentE. coli (Invitrogen), and the reaction mixture is incubated on ice for10 min, heat-shocked to 42° C. (30 sec) and incubated on ice for another2 min. A total of 250 μl SOC medium (Invitrogen) is then added to thecells which are then shaken at 37° C. (1 hr). This transformationreaction is then diluted 1:100 or 1:10 using SOC medium. A total of 50μl of each is spread on LB plates containing 12.5 μmol/ml ampicillinthat are incubated overnight at 37° C. A representative number ofcolonies are randomly chosen to inoculate a corresponding number of 5 mlovernight LB cultures in the presence of 12.5 μmol/ml ampicillin.Plasmids extracted from each liquid culture are analyzed by automatedDNA sequencing using the fluorescence-based dideoxy chain terminationreaction (Genewiz, North Brunswick, N.J.) to determine if they containthe correct p53 cDNA reading frame associated with a stop codon and astart codon embedded in the Kozak sequence.

The same procedure is followed for preparation of a plasmid encoding ascrambled sequence comprising the amino acids of the p53 peptidesequence of SLH-1, with start, stop codons, and restriction enzyme sitesdenoted as above.

In addition to this plasmid, two other plasmids encoding proteins thatserve as controls are prepared: full-length HDM-2 without the CAAXsequence, called pHDM2 and HDM-2-CVVK, that lacks amino acid residues1-109 that constitute the binding site for p53 and for SLH-1, calledpdell-109-HDM2-CVVK. All plasmids are prepared at Origene. The followingprimers are employed to construct the DNA sequences encoding each of theHDM-2 proteins. For these sequences all nuclease sites are given inbold; the start (ATG) and stop (TTA) codons are italicized; and thecarboxyl terminal codons for the membrane-localization signal sequence,CVVK (CAAX box), are underlined: Full-length HDM-2:

(SEQ ID NO: 29) CTACAGCGATCGCCATGGTGAGGAGCAGGCAAATGTGC (+strand),(SEQ ID NO: 30) ACGAGACGCGTGGGGAAATAAGTTAGCACAATCATTTG (−strand);Full-Length HDM-2 with C-terminal CVVK membrane-attaching CAAX sequence

(SEQ ID NO: 31) CTACAGCGATCGCCATGGTGAGGAGCAGGCAAATGTGC (+strand)(SEQ ID NO: 32) GCGTACGCGT TTA CATAATTACACACTTGGGGAAATAAGTTAGCACAATCATTTGG (−strand);HDM-2-CVVK with residues 1-109 deleted

(SEQ ID NO: 33) CTACAGCGATCGC CATCTACAGGAACTTGGTAGTAGTC (+STRAND)(SEQ ID NO: 34) GCGTACGCGT TTA CATAATTACACACTTGGGGAAATAAGTTAGCACAATCATTTGG (−STRAND)

After digestion with the cloning restriction enzyme Sgf-I and Mlu I, thePCR products are cloned into the Origene Precision Shuttle plasmidpCMV6-AN-GFP with an N-terminal fused GFP-tag. Final constructs aresequenced with VP1.5 5′ GGACTTTCCAAAATGTCG 3′ (SEQ ID NO: 35) and XL395′ ATTAGGACAAGGCTGGTGGG 3′ (SEQ ID NO: 36) primers.

Transfection of HDM-2 Constructs into Untransformed Cells

The plasmids that are constructed as described above are transfectedinto untransformed MCF-10-2A cells using transfection methods known inthe art, for example in Bowne et al. supra. The transfection efficiencyis evaluated by analyzing the GFP fluorescence at 480 nm.

Cell Lines

The following cell lines are obtained from the American Type CultureCollection (Manassas, Va.): MIA-PaCa-2 (human pancreatic cancer), MCF-7(human breast cancer), A2058 (human melanoma), MCF-10-2A (normal humanbreast epithelial cells). Ag13145 cells (primary human fibroblasts, 46chromosome, XY) are obtained from the Coriell Institute for MedicalResearch (Camden, N.J.) and cultured in DMEM supplemented with 10% fetalbovine serum (FBS; Atlanta Biologicals, Atlanta, Ga.). In addition, twocell lines, i.e., BMRPA1, a normal rat pancreatic acinar cell line, andits k-ras-transformed counterpart pancreatic cancer cell line, calledTUC-3 (Kanovsky et al. 2001 Proc Natl Acad Sci USA, 98:12438-12443),both of which were grown in cRPMI medium are useful according to theinvention.

The human chronic myeloid K562 leukemia cell line, that lacks p53expression, was obtained from American Type Culture Collection (ATCC,Manasass, Va.). Cell cultures were maintained in RPMI-1640 media(Sigma-Aldrich, St. Louis, Mo.) supplemented with 10% fetal bovine serum(FBS) and 1% penicillin/streptomycin (P/S) [100 U/100 μg/ml] at 5% CO₂in a humidified incubator at 37° C.

Murine leukocytes. Mouse peritoneal macrophages are isolated by astandard procedure (Zhang et al. 2008 Curr Protoc Immunol Chapter Unit14-1 (DOI: 10.1002/0471142735.im1401s83)) from WT C57B16/j and SphK-1 KOmice. To isolate peritoneal macrophages, the mice are injected with 4%thioglycolate medium intraperitoneally, and peritoneal lavage fluid iscollected 72 h post-injection. Next, the CD11b+ macrophages inperitoneal lavage are purified by MACS based positive selection methodusing CD11b+ magnetic micro beads (Miltenyi Biotech, Auburn, Calif.).Peritoneal macrophages per well are cultured in RPMI-1640 medium(Sigma-Aldrich, St. Louis, Mo.) and incubated at 37° C. in 5% CO₂.

Expression of the p53 Peptide of SLH-1

For protein analyses, and detection of apoptosis, 2×10⁶ cells (cancerand non-cancerous cells) are seeded into 10 cm diameter tissue cultureplates and transfected with DNA: Lipofectamine 2000 proportionallyadjusted to the increased area. When the cell density reaches 90-100%,the cells of the experimental and sham-transfected group are detachedusing trypsin and plated into four new plates in which they are allowedto grow in complete medium. At defined time points cells are releasedfrom adherence with 10 mM EDTA in PBS and are lysed in lysing buffer [1%Triton X-100 in 0.05 M Tris-HCl (pH 8.0), 0.15 mM NaCl, 0.02% Na azide,0.01 mg/ml phenylmethylsulfonylfluoride (PMSF), and 0.001 mg/mlAprotinin]. Protein equivalents of 10⁶ cells, i.e., about 30 μg/lane,are then subjected to SDS-PAGE using 10% Tris-HCl gels and, in someexperiments, 16% Tricine Peptide Gels (Biorad, Hercules, Calif.) todetect SLH-1 and the p53 portion of SLH-1. The separated proteins arethen electrophoretically transferred to nitrocellulose membranesfollowed by immunoblotting with the mAb DO-1 to p53 AA residues 11-25,and with mAb B-2 to GFP (each at 1 μg-2.5 μg/ml blotting buffer),respectively. After washing non-reacted mAbs from the membranes, themembranes are incubated (1 h) with a second enzyme-labeled antibody fromthe ECL chemiluminescence kit (Amersham, Piscataway, N.J.) to detect thepresence of p53 and the p53 peptide of SLH-1. A time course of GFPexpression is performed in both cancer and non-cancerous cells todetermine when the highest levels of GFP expression occurs.Semi-quantitation of immunoblotting results is performed by measuringluminosity of bands in a single scanned developed x-ray film, using thehistogram option of Adobe Photoshop 5.5. Background is ascertained bymeasuring average luminosity of 5 areas of the film outside the blottingregion. Opacity of each band is calculated by the equation,Opacity=255-Luminosity-background.

Incubation of Cells with SLH-1

Duplicate sets of 6×10⁶ cancer cells are incubated with differentconcentrations of SLH-1, i.e., 5, 10, 20, 40, 80 and 160 μmol/L.Duplicate control experiments are also performed in which 6×10⁶ cancercells are incubated with the control, PNC-29, present at a concentrationof 75 μmol/L. After the cells have been allowed to adhere to the tissueculture dish (TCD) for 24 hours the medium is removed from each TCD, andnew medium containing the same concentration of peptide or no peptide isadded. Medium from each TCD is removed every 24 h, and fresh medium withits respective peptide at the appropriate concentration is added. Cellsare inspected daily for changes in cell growth, morphology, andviability. At the end of each day over a five-day period, duplicate cellcounts are performed for each incubation using the trypan blue exclusionmethod. In addition, cell viability is also determined by a3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT)assay according to the manufacturers' instructions (Promega Corporation,Madison, Wis., USA).

Incubation of Peptides with BMRPA1 Cells

These control cells are untransformed rat pancreatic acinar cells.Duplicate 5-day incubations are performed on 6×10⁶ cells in threecircumstances: with no peptide, with SLH-1 at 75 μmol/ml, and withPNC-29 at 75 μmol/ml. Cells are assessed for viability and morphologyover this time period. At the end of 5 days, cell counts are performedusing the trypan blue exclusion method.

Immunocytochemistry for Annexin V-Binding to Phosphatidyl Serine

To determine whether any of the transfected plasmids induce apoptosis,the cells are evaluated to determine whether the cells containphosphatidyl serine in the inner cell membrane, identified as binding toannexin-V, as a marker for apoptosis. Cells (5×10⁵) are seeded in 6-welltissue culture plates 24 h prior to transfection in antibiotic-freemedium. Cells are then either transfected with empty vector, vectorencoding the p53 portion of SLH-1 or a vector encoding the scrambledpeptide or are left untreated. At predetermined time-pointspost-transfection, the cells are released using 0.5.times Trypsin-EDTA,collected and processed as described in the manufacturer's instructionsof the Annexin V-Biotin Apoptosis Detection Kit (CalBioChem, La Jolla,Calif). The stained cells are resuspended in antifade (Molecular Probes,OR), mounted on glass slides under a glass coverslip and evaluated forred (TRITC) and green (GFP) fluorescence using confocal microscopy asdescribed above.

Evaluation of Cells Treated with SLH-1 for Caspase as a Marker forApoptosis and LDH Release as a Marker for Necrosis

Cancer cells from culture plates at 18, 44, 66 and 90 h time points arelysed in situ in cell lysis buffer [1% Triton X-100 in 0.05 M Tris-HCl(pH 8.0), 0.15 mM NaCl, 0.02% Na azide, 0.1 mg/mlphenylmethylsulfonylfluoride (PMSF), and 0.001 mg/ml Aprotinin]. Lysatesare subjected to 10% SDS-PAGE followed by electrotransfer tonitrocellulose and immunoblotting with antibodies to GFP and p53 (SantaCruz Biotechnology, Santa Cruz, Calif.). Antibody-labeled proteins areidentified by chemiluminescence using ECL methodology (Amersham). Assaysfor elevated caspase expression are performed using the Clontech (PaloAlto, Calif.) for caspase (CPP32) activity. As a positive control forthe caspase activity assay, replicate samples of cancer cells areincubated with tumor necrosis factor (TNF) (Sigma, St. Louis, Mo.) at aconcentration of 10 ng/ml for 24 h. In addition, to detect ifsignificant cell necrosis occurs, the CytoTox96 assay is used (Promega,Madison, Wis.) to measure LDH released into the cell culture medium.

Lactate dehydrogenase (LDH) assay using the LDH Cytotoxicity Assay(Promega, Madison, Wis.); caspase assay for apoptosis [positive control:2×10⁴ cells treated with staurosporine (Sigma, St Louis, Mo.) (45μg/ml)]; and MTT cell viability assay are all performed as described inDo et al. 2003 Oncogene, 22:1431-1444; Pincus et al. 2007 ResearchAdvances in Cancer, ed Mohan R (Global Research Network Publishers,Kerala, India), pp 65-90; and Bowne et al. 2008 Ann Surg Oncol,15:3588-3600). Protein concentrations are determined using the Bradfordassay (Pierce, Rockford, Ill.).

Electron Micropscopy of Cancer Cells Treated with SLH-1

Time-lapse electron microscopy (EM) is used to examine theultrastructural features of cell death. Cancer cells are grown for 24 hon Thermanox cover slips (Lux Scientific), and then treated with 25 μmolof SLH-1 for 1 and 15 min, along with a corresponding control groupwithout peptide. The cells are washed with PBS solution and then fixedwith 2.5% gluteraldehyde-PBS. The fixed cultures are rinsed in a 0.1 Mphosphate buffer (pH 7.3), post fixed in 2% (0.08 m) osmiumtetroxide-PBS (pH 7.3), dehydrated in a graded series of ethanol andpropylene oxide and embedded in Epon 812. Sections are cut at 700 ANG.,stained with uranyl acetate and lead citrate and examined with a JeolJEM 1010 Electron Microscope.

Blotting of Cancer Cell Lysates for p53 and Waf^(P21), a Target forActivated p53

Cell lysates are prepared as described above and are subjected toimmunoblotting with either DO-1 antibody described above for expressionof the p53 peptide, a (Ab-6) monoclonal anti-p53 antibody (Calbiochem)or with polyclonal anti-waf^(p21) antibody (Santa Cruz Biotechnology,Santa Cruz, Calif.) (1:2000 dilution) as described above. As a control,actin is detected using anti-actin polyclonal antibody (Santa CruzBiotechnology).

Western Blots

Lysates of 2×10⁶ cells are either used directly or employed forpreparation of purified plasma membranes. To assure that the finalpreparations contains plasma membranes, samples are immunoblotted formembrane β-catenin and by transmission electron microscopy. Blotting ofboth fractions for H(M)DM-2 is performed in a manner similar to thatdescribed previously. Anti-HDM-2 polyclonal antibody (Santa CruzBiotechnology, Santa Cruz, Calif.) is used at a dilution of 1:4,000.Secondary antibody is HRP-conjugated donkey anti-mouse IgG (HRP-anti-MIgG) (Jackson ImmunoResearch, West Grove, Pa.) used at a dilution of1:1,000 in 0.1% milk in TBS-T. Immun-Star HRP Peroxide Buffer+Immun-StarHRP Luminol/Enhancer (ratio 1:1) (BioRad) is added to the nitrocellulosemembranes.

Alternatively, after 48 h of treatment with peptides, the cells areharvested from culture plates and lysed in cell lysis buffer containing1% Triton X-100 in 0.05 M Tris-HCL (pH 8.0), 0.15 mM NaCl, 0.02% sodiumazide, 0.1 mg/ml phenylmethylsulfonylfluoride (PMSF), and 0.001 mg/mlAprotinin. Lysates are subjected to 10% SDS-PAGE followed byelectrotransfer to PVDF membrane (Invitrogen, Carlsbad, Calif.) andimmunoblotting with DO-1 monoclonal anti-p53 (Santa Cruz BiotechnologyInc., Dallas Tex.) [1:2000] and anti-actin (Sigma-Aldrich, St. Louis,Mo.) [1:4000] antibodies. Antibody-labeled proteins are identified bychemiluminescence using Super Signal West Pico ChemiluminescentSubstrate (Thermo Scientific Rockford, Ill.). Bradford Dye Reagent(Bio-Rad Laboratories, Hercules, Calif.) is used to determine theprotein concentration.

Immunoprecipitation Experiments

To determine if fluorophore-labeled SLH-1 binds to HDM-2 in cancer cellstreated with this peptide, double-fluorophore-labeled SLH-1 (50 μg/ml)is incubated with 1×10⁶ MIA-PaCa-2 cells for 4 hr. The cells are thenlysed (Kanovsky et al. supra; Do et al. supra; Pincus et al. supra; andBowne et al. supra). Anti-HDM-2 polyclonal antibody (Santa CruzBiotechnology, Santa Cruz, Calif.), and 500 μg protein in lysate samplesare subjected to IP with 0.5 μg anti-MDM2 mouse monoclonal antibody(D-7, Santa Cruz Biotechnology), 2 μg biotinylated horse antimouse IgG(Pierce) and 30 μl 50% suspension of UltraLink-immobilized NeutrAvidinBiotin-Binding Protein beads. The samples are then electrophoresed in12.5% PAAG gel (BioRad). The position of the fluorescent peptide isanalyzed in the gel with Kodak Image Station 2000R. The proteins aretransferred to a PVDF membrane, immunobloted with polyclonal anti-MDM2antibody (Santa Cruz, N-20), and developed with ECL (Pierce).

Colocalization Experiments and Confocal Microscopy

These experiments are performed on any of the cancer cell linesdisclosed herein, or any available cancer cell lines. Cells grown onglass cover slips to 50-60% density are treated for up to 15 min at 37°C. in a humidified 5% CO₂-95% air incubator chamber with SLH-1 or PNC-29(control) at 50 μg/ml incubation medium and are then washed and fixed in3% paraformaldehyde in PBS (pH 7.2) supplemented with 0.01%glutaraldehyde for 1.5 h followed by extensive washing and transfer intoPBS for storage until mounting on glass slides for microscopy. Freealdehyde groups are quenched by incubating cells with glycine (0.2 M)and sodium borohydride (75 mM) followed by washing in PBS. Cells arethen stained (direct staining) for 2 h, 4° C., with fluorescein-labeledmouse monoclonal antibody against p53 [FITC-mAbα-p53 (DO-1)] (5 μg/ml)and rhodamine-labeled (TRITC-) mAbα-against H/R/MDM-2 (5 μg/ml) (bothlabeled antibodies, Pierce) in 1% FBS-PBS. After removal of nonreactiveAb and extensive washing, the cover slips are mounted on glass slidesover Prolong Gold Antifade (Molecular Probes, Invitrogen, Carlsbad,Calif.) and examined with a laser-equipped Olympus Confocal microscope1×76 (Olympus America Inc, Center Valley, Pa.). Results are digitallyrecorded. The colocalization of the two antibodies is confirmed byoverlapping green (anti-SLH-1) and red (antiH/R/MDM-2) fluorescentlabels that produce yellow fluorescence (combined green and redfluorescence).

Dose Response Experiments

LDH assays are performed on different cancer cell lines (n=3-5) that areincubated for 30 min with SLH-1 over a concentration range of 10 μg·ml-1mg/ml as described previously (Do et al. supra; Pincus et al. supra; andBowne et al. supra).

Treatment of Transfected Cells with SLH-1

Transfected cells in media are incubated at 37° C. with 5% CO₂ for 24 h,at which time they are treated with SLH-1 peptide (sonicated brieflyprior to addition) such that the final concentration is 300 μg/ml.Samples are assayed for LDH, MTT, and caspase. In addition, samples areprocessed for confocal microscopy as described above except for thefollowing modification. Because the cells contain GFP, to localizeSLH-1, it is necessary to use another fluorescent probe other thangreen-fluorescent FITC-labeled DO1 antibody. Cells are incubated withunlabeled DO1 and anti-HDM-2 as described above. The cells are thenwashed and incubated with Alexa Fluor 647 goat antimouse IgG (1:200)(against DO1 mouse) (Invitrogen-Molecular Probe, Eugene, Oreg.) andTAMRA-labeled goat antirabbit IgG (1:200) (against anti-HDM-2 rabbitpolyclonal IgG) (Sigma, St. Louis, Mo.). The cells are processed forconfocal microscopy, and the membrane fractions and whole cell lysatesare blotted for either HDM-2 or actin [rabbit anti-actin-42 polyclonalantibody (1:5000)] (Sigma).

Alternatively, after reaching the desired confluence cells are incubatedwith different concentrations of SLH-1 or PNC-29 (i.e., 25, 50, 75, 100μM) in tissue culture dishes (TCD) for 24 h. For 48 h treatments, thesame peptide concentration is added on the second day as describedpreviously (Karnovsky et al. supra). Cells are inspected daily forchanges in cell growth, morphology, and viability. At the end of a 48 hperiod, cell viability is determined by MTT(3-[4,5-dimethylthiazol-2yl]-2,5-diphenyl tetrazolium bromide) assayaccording to the manufacturer's instruction (Promega, Madison, Wis.)

Confocal Microscopy

Cells (2×10⁶) are maintained in RPMI-1640 Media (Sigma-Aldrich, St.Louis, Mo.) supplemented with 10% FBS and 1% P/S [100 U/100 μg/ml] andare treated with SLH-1 (100 μg/ml) or PNC-29 (100 μg/ml) for 1 h in 5%CO₂ humidified incubator at 37° C. All samples are then washedrepeatedly with PBS. The cells are fixed with 4% formaldehyde in PBS for5 minutes at room temperature and blocked with PBS containing 5% bovineserum albumin (BSA) and 0.3% Triton x-100 for 1 h on a shaker at roomtemperature. An antibody mixture of DO1 anti-p53 [1:250] (Santa CruzBiotechnology Inc., Dallas Tex.) that recognizes p53 aa 11-25 thatoverlaps the modified 12-26 p53 sequence of SLH-1 and anti-HDM-2polyclonal antibody (N-20, sc813, Santa Cruz Biotechnology, Dallas,Tex.) [1:250] in 1% BSA and 0.3% Triton x-100 containing PBS is added tocells overnight on a rotator at 4° C. The cells are then washedextensively with PBS and treated with a mixture of secondary antibodies,the polyclonal goat to mouse greenfluorophore (DyLight® 488) [1:200](Abcam Inc., Cambridge, Mass.) and polyclonal goat to rabbit redfluorophore (DyLight® 650) [1:200] (Abcam Inc., Cambridge, Mass.) in PBScontaining 1% BSA and 0.3% Triton x-100 overnight on a rotator at 4° C.After incubation cells are washed repeatedly with PBS and visualized onglass slides under the Olympus Fluoview FV1000 confocal microscope(Olympus America INC, Center Valley, Pa.) using a 60×NA 1.42 PLAPON oilobjective. Sequential image recording has been applied to avoid spectraloverlap. Image analysis is performed using Olympus FV10-ASW 1.7 Viewersoftware.

Compositions useful according to the invention include SLH-1 incombination with additional peptides and/or non-peptide materials whichmay desirably have an HDM-2 affinity or binding site and may be used inconjunction with the MRP. Hybrid materials containing peptide andnon-peptide components, along with wholly non-peptide materials may beused with one or more of the methods of the present invention. Thesynthesis of one or more of the compounds may be subsequently followedby purification, as is commonly done in the art. The compoundssynthesized are preferably in purified form to be used as the compoundand with the methods of the present invention. Thus, the presentinvention contemplates the use of peptide as well as non-peptidematerials, and combinations thereof, to cause selective necrosis tocancer cells, in accordance with the present invention.

It should be readily understood and appreciated that each of theelements and features of the present invention discussed with oneembodiment may be similarly employed with other embodiments disclosedherein, and this discussion is by no means deemed limiting to thevarious additional permutations that may be employed, for example, withthe methods presented herein.

Unless defined otherwise, all technical and scientific terms used hereinhave the meaning commonly understood by a person skilled in the art towhich this invention belongs. The following references provide one ofskill with a general definition of many of the terms used in thisinvention: Singleton et al., Dictionary of Microbiology and MolecularBiology (2nd ed. 1994); The Cambridge Dictionary of Science andTechnology (Walker ed., 1988); The Glossary of Genetics, 5th Ed., R.Rieger et al. (eds.), Springer Verlag (1991); and Hale & Marham, TheHarper Collins Dictionary of Biology (1991). As used herein, thefollowing terms have the meanings ascribed to them below, unlessspecified otherwise.

The present invention is described by reference to the followingExamples, which are offered by way of illustration and are not intendedto limit the invention in any manner Standard techniques well known inthe art or the techniques specifically described below are utilized.

EXAMPLES Example 1 Cytotoxicity of SLH-1 for Cancer Cells

SLH-1 is incubated with 6×10⁶ cancer cells, for example, Mia-PaCa-2cells, for 5 days at concentrations ranging from 12.5-75 μmol/ml. Atdifferent timepoints following treatment the cells are assessed forsigns of necrosis, for example, membrane blebbing and disruption andformation of cell clumps coalescing into aggregates of cellular debris.Percent cell death is determined by measuring trypan blue dye uptake.Cancer cells are also treated with the negative control peptide PNC-29and, separately, a peptide having only the p53 derived portion of SLH-1that cannot traverse the cell membrane due to the lack of the MRPpeptide, administered at the same concentration as the test peptide. Theeffect of these control peptides on cellular growth, morphology andviability is determined.

As an additional control, SLH-1 is combined with untransformed BMRPA1acinar cells and with the untransformed breast epithelial cell line,MCF-10-2A at a concentration ranging from 12.5-75 μmol/ml to determineif the peptide is lethal to normal cell growth.

Example 2 Markers for Necrosis and Apoptosis in Cancer Cells Treatedwith SLH-1

Cell death can occur by either necrosis or apoptosis. p53-targetingtreatments typically cause cell death through apoptosis. Necrosis is notgenetically controlled, while apoptosis is genetically controlled.Apoptosis is the deliberate cellular response to specific environmentaland developmental stimuli or programmed cell death. Cells undergoingapoptosis exhibit cell shrinkage, membrane blebbing, chromatincondensation and fragmentation. Necrosis involves the destruction ofcytoplasmic organelles and a loss of plasma membrane integrity. Thoughapoptosis does not have the inflammation which results when cancer cellsdie through necrosis, p53 targeting treatments fail to treat thosecancers that do not exhibit p53, or, through mutations, exhibit aninactive p53 form that is unresponsive to p53 targeted treatments. Afterthe DNA damage in the caspase enzyme pathway, there are a series ofevents which occur that involve calcium activation and calpain enzymeswhich further leads to other cellular changes and regulation ofcytoplasmic enzymes. During p53-dependent apoptosis, there is asequential expression of annexin V-binding membrane phospho-Serine, Baxwaf^(p21), and caspases; these proteins are used as markers forp53-dependent apoptosis.

A major difference between necrosis and apoptosis in vivo is thecomplete elimination of the apoptotic cell before an inflammatoryresponse is seen. Necrosis usually causes inflammation. Though apoptosiscan be thought of as a clean and neat process, the p53 targetingtreatments do not result in apoptosis in all types of cancer cases.Though necrosis may typically cause an inflammatory response to atreatment site directed at targeting HDM-2, HDM-2-targeting treatmentsare more effective against various forms of cancer, including thosewhere p53 is not present in the cancer cells, or where p53 is in amutated or an inactive form.

To determine if SLH-1 induces cell death by necrosis or by apoptosis,the expression of LDH and caspase in cancer cells treated with SLH-1 isdetermined as described above. Early release of LDH (elevation of LDHlevels) indicates necrosis Elevated levels of caspase are indicative ofapoptosis

Electron micrographs of cancer cells or non-cancerous cells treated withSLH-1 and untreated cells are performed to determine if the cellsexhibit lysis of their plasma membranes or have intact plasma membranes.Lysis of plasma membranes is characteristic of tumor cell necrosis.

Example 3

Transfection of Cancer Cells with a Plasmid that Encodes the p53Sequence of SLH-1

Cancer cells and non-cancerous cells are transfected with empty vectoror a green fluorescent protein encoding vector encoding the p53 sequenceof SLH-1. After 2 hours post-transfection, cell counts are performed onslides using light microscopy followed by counting the number of cellsexhibiting green fluorescence from GFP (Green Fluorescent Protein).Morphological examination of transfected cells, as visualized byinverted light microscopy is also performed to identify cells that arenephrotic or apoptotic.

Cancer cells, for example MiaPaCa-2 cells expressing GFP that aretransfected with empty vector a vector expressing the p53 sequence ofSLH-1 are lysed and blotted for p53, waf^(p21), a protein that isinduced by a p53-dependent pathway, and the p53 17-26 peptide itself. Inthese experiments, the DO-1 anti-p53 antibody that recognizes adeterminant that contains residues 17-26 of p53 is used. In addition,caspase activity in these cells is measured. For comparison, the sameset of experiments are performed on cancer cells treated with 75 μmol/mlof SLH-1. Caspase activity and p53^(waf21) levels are determined. Forcontrols, actin is blotted for.

In the early stages of apoptosis, phosphatidyl serine (PS), normallypresent in the inner leaflet of the bilayer membrane of intact cells, isfound on the external plasma membrane of cells undergoing apoptosis.Annexin V binds PS and can be located by a probe that carries the redfluorescent TRITC probe. Cells that are transfected with the p53sequence of SLH-1 or control vector are processed for staining withAnnexin V-biotin followed by streptavidin-TRITC and examined by confocalmicroscopy. Control experiments with BMRPA1 control cells transfectedwith a vector expressing the p53 sequence of SLH-1 or a control vectorare also performed and these cells are analyzed for expression ofAnnexin V.

Example 4

Transfection of Cells with pTracer-SV40 Plasmid Encoding Only the p53Sequence of SLH-1

To define the role of the MRP definitively, the effects of the p53peptide of SLH-1 itself on tumor cell growth, i.e., whether even withoutthe MRP, it could induce tumor cell necrosis, are determined. The p53peptide of SLH-1 is introduced into cancer cells via transfection usingthe pTracer-SV40 plasmid that constitutively expresses this peptide. Theexpression of markers for apoptosis and necrosis in the transfectedcells are measured and compared with the levels of these markers inreplicate samples of cancer cells treated with SLH-1. These experimentsare also performed in BMRPA1 control cells.

Example 5

Induction of Apoptosis in Cancer Cells Treated with the p53 Peptide ofSLH-1

Experiments are performed in cancer cells expressing the p53 peptide ofSLH-1. To determine if apoptosis is occurring, the expression ofWaf^(p21) is determined. Apoptosis is indicated by increased levels ofWaf^(p21). The level of caspase is also determined. An increase in thelevel of caspase is indicative of apoptosis. The level of expression ofannexin-V-binding phosphatidyl serine in the membranes, a known earlyphenomenon in apoptosis, is determined in cancer cells transfected withthe p53 peptide of SLH-1 as well as cells transfected with empty vector.The level of LDH release is also measured. It is expected that anincrease in the release of LDH will occur if the peptide induces tumorcell necrosis.

Example 6 In Vivo Analysis of SLH-1 Activity

Nu/Nu mice (Harlan Laboratories, Indianapolis, Ind., n=10) weighing20-22 g, are xenotransplanted subcutaneously (s.c.) with live pancreaticcarcinoma cells BMRPA1.TUC-3 (1×10⁶ cells/mouse) in the left hindregion. Tumors are allowed to develop and grow.

After tumor formation has occurred, the mice are separated into threegroups. Each group is implanted s.c. with Alzet™ osmotic pumps todeliver in a constant rate and over a defined period of 14 days a totalvolume of 0.095 ml volume of normal saline containing the peptide at aconcentration of 20 mg/mouse. One group of mice receives SLH-1 (SEQ IDNO:2) fused at its carboxy terminal end to the penetratin leadersequence (SEQ ID NO:3) and the other group of mice receives PNC-29, acontrol peptide of similar size, having the following amino acidsequence: MPFSTGKRIMLGE (SEQ ID NO: 37). A third group of mice does notreceive peptide. The pumps are filled according to the manufacturersguidelines and under sterile conditions The pumps are implanted s.c. onthe left flank of the anaesthetized mice by creating a pocket underneaththe mouse skin into which the tiny pumps are inserted. Each pocket isclosed with a simple suture. From their inside chamber the pumps delivercontinuously 0.25 μl/hr into each mouse. The mice are observed untilthey have recovered from the surgery and are then returned to theisolation ward of the animal facility. Since the animals are Nu/Nu miceand, thus, immuno-compromised they are highly susceptible when exposedto pathogens. Surgery and all preceding and post-surgical treatments aretherefore performed in a sterile hood

Alternatively, using the same methodology as described above, livepancreatic carcinoma cells BMRPA1.TUC-3 (1×10⁶ cells/mouse) aretransplanted to the peritoneal cavity of five mice and pumps are placedin the right shoulder region at the same time of tumor celltransplantation.

All patents and publications mentioned in the specification areindicative of the levels of skill of those skilled in the art to whichthe invention pertains. All references cited in this disclosure areincorporated by reference.

One skilled in the art would readily appreciate that the presentinvention is well adapted to carry out the objects and obtain the endsand advantages mentioned, as well as those inherent therein. The methodsand compositions described herein as presently representative ofpreferred embodiments are exemplary and are not intended as limitationson the scope of the invention. Changes therein and other uses will occurto those skilled in the art, which are encompassed within the spirit ofthe invention, are defined by the scope of the claims.

It will be readily apparent to one skilled in the art that varyingsubstitutions and modifications can be made to the invention disclosedherein without departing from the scope and spirit of the invention.Thus, such additional embodiments are within the scope of the presentinvention and the following claims.

The invention illustratively described herein suitably can be practicedin the absence of any element or elements, limitation or limitationsthat are not specifically disclosed herein. Thus, for example, in eachinstance herein any of the terms “comprising”, “consisting essentiallyof”, and “consisting of” may be replaced with either of the other twoterms. The terms and expressions which have been employed are used asterms of description and not of limitation, and there is no intentionthat in the use of such terms and expressions of excluding anyequivalents of the features shown and described or portions thereof, butit is recognized that various modifications are possible within thescope of the invention claimed. Thus, it should be understood thatalthough the present invention has been specifically disclosed bypreferred embodiments, optional features, modification and variation ofthe concepts herein disclosed may be resorted to by those skilled in theart, and that such modifications and variations are considered to bewithin the scope of this invention as defined by the description and theappended claims.

In addition, where features or aspects of the invention are described interms of Markush groups or other grouping of alternatives, those skilledin the art will recognize that the invention is also thereby describedin terms of any individual member or subgroup of members of the Markushgroup or other group.

The use of the terms “a” and “an” and “the” and similar referents in thecontext of describing the invention (especially in the context of thefollowing claims) are to be construed to cover both the singular and theplural, unless otherwise indicated herein or clearly contradicted bycontext. The terms “comprising,” “having,” “including,” and “containing”are to be construed as open-ended terms (i.e., meaning “including, butnot limited to,”) unless otherwise noted. Recitation of ranges of valuesherein are merely intended to serve as a shorthand method of referringindividually to each separate value falling within the range, unlessotherwise indicated herein, and each separate value is incorporated intothe specification as if it were individually recited herein. All methodsdescribed herein can be performed in any suitable order unless otherwiseindicated herein or otherwise clearly contradicted by context. The useof any and all examples, or exemplary language (e.g., “such as”)provided herein, is intended merely to better illuminate the inventionand does not pose a limitation on the scope of the invention unlessotherwise claimed. No language in the specification should be construedas indicating any non-claimed element as essential to the practice ofthe invention.

Embodiments of this invention are described herein, including the bestmode known to the inventors for carrying out the invention. Variationsof those embodiments may become apparent to those of ordinary skill inthe art upon reading the foregoing description. The inventors expectskilled artisans to employ such variations as appropriate, and theinventors intend for the invention to be practiced otherwise than asspecifically described herein. Accordingly, this invention includes allmodifications and equivalents of the subject matter recited in theclaims appended hereto as permitted by applicable law. Moreover, anycombination of the above-described elements in all possible variationsthereof is encompassed by the invention unless otherwise indicatedherein or otherwise clearly contradicted by context.

What is claimed is:
 1. A method of treating cancer in a cell, comprisingcontacting the cell with a peptide comprising the amino acid sequencePPLSQTSFAEYWNLLSP (SEQ ID NO: 2) or a peptide comprising the amino acidsequence:H-Pro-Pro-Leu-Ser-Gln-Thr-Ser-Phe-Ala-Glu-Tyr-Trp-Asn-Leu-Leu-Ser-Pro-Lys-Lys-Trp-Lys-Met-Arg-Arg-Asn-Gln-Phe-Trp-Val-Lys-Val-Gln-Arg-Gly-OH(SEQ ID NO: 3).
 2. The method of claim 1 wherein the cancer is selectedfrom the group consisting of: astrocytoma, bladder cancer, brain cancer,breast cancer, cervical cancer, colorectal cancer, endometrial cancer,gastric cancer, lung cancer, melanoma, leukemia, lymphoma, ovariancancer, pancreatic cancer, prostate cancer, renal carcinoma, sarcoma,thyroid cancer, glioblastoma, multiple myeloma, myelodysplasticsyndrome, mesothelioma, acute myeloid leukemia, childhood leukemia,chronic myeloid leukemia, myelodysplastic syndrome, Hodgkin's lymphoma,and Polycythemia Vera.
 3. The method of claim 1 wherein the peptide isat an amount between 1.5 μmol/ml and 75 μmol/ml.
 4. The method of claim1, wherein the peptide further comprises a membrane penetrating aminoacid sequence.
 5. The method of claim 4, wherein said membranepenetrating sequence forms the carboxy terminal sequence of saidpeptide.
 6. The method of claim 4, wherein said membrane penetratingsequence comprises the amino acid sequence: KKWKMRRNQFWVKVQRG (SEQ IDNO: 4).